Recovery of lignin and water soluble sugars from plant materials

ABSTRACT

In one aspect, a process for treating woody plant material is provided, the process involving contacting the plant material with a continuous flow of an aqueous ethanol solution at elevated temperature and pressure under conditions that promote extraction of ethanol-soluble lignin from the plant material and retention of hemicellulose sugars, xylose and cellulose in the treated plant material solids. In another aspect, a process for extracting hemicellulose sugars from lignin-depleted plant material solids is provided, the process involving contacting lignin-depleted plant material with water at elevated temperature and pressure under conditions that promote extraction of hemicellulose sugars from the plant material; and recovering hemicellulose sugars from the liquid mixture.

REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.13/189,388, filed Jul. 22, 2011, which is a divisional of U.S. patentapplication Ser. No. 12/638,862 filed Dec. 15, 2009, which is acontinuation of U.S. patent application Ser. No. 11/745,993 filed May 8,2007, now U.S. Pat. No. 7,649,086 issued Jan. 19, 2010, which claimspriority to U.S. Provisional Patent Application No. 60/746,682 filed May8, 2006 and 60/869,057 filed Dec. 7, 2006.

FIELD OF THE INVENTION

The present invention provides methods and apparatus for the productionof biofuel from plant materials. More specifically, the presentinvention provides an integrated process for the production of plantbiomass such as Salix spp. and its conversion to ethanol and othervaluable products.

BACKGROUND

Woody biomass can be employed as a sustainable source of energy and is avaluable alternative to fossil fuels. More specifically, the biorefiningof lignocellulosic material into fuel ethanol and lignin materials hasthe potential to displace a portion of petrol and oil based materials.It is likely that, with the depletion of global oil reserves andincreasing awareness of the environmental and national security issuesassociated with dependence on fossil fuel, biomass will become a keyresource for the production of transport fuel in much of the world.

The conversion of lignocellulosic biomass into fuel ethanol may offerthe ideal solution given the rapid growth of short rotation crops suchas shrub willow (Salix spp.). Two of the main components of wood,cellulose and hemicellulose, are polymers of simple sugars that can beconverted into ethanol and/or other chemicals by fermentation. Thisethanol can be used as a transportation fuel either on its own or as anethanol-gasoline blend. Ethanol-gasoline blends of up to 10% ethanol canbe used without any engine modification or loss in engine performance(Hunt, V. D. (1981) The Gasohol Handbook, New York, Industrial Press).Lignin, the third main component of wood, is a potential raw materialfor the production of plastics, adhesives and resins (Lora and Glasser(2002) J. Polymers Environ. 10:39-47). The use of lignin in high valueproducts, rather than as boiler fuel, will off-set the high coststraditionally associated with the processing of wood and production ofethanol.

Willow biomass plantations can be easily and efficiently establishedfrom dormant stem cuttings using mechanical systems. Shrub willowsrespond to coppicing after the first growing season by prolificproduction of new stem growth in the second growing season. Above groundwoody biomass is harvested during the dormant season. During the springfollowing each harvest, the remaining portion of the willow plant, knownas the stool, responds by producing numerous new stems, initiating a newcycle of growth that can be harvested in another two to four years. Thiscycle can be repeated for six to eight harvests before the stools needto be replaced.

Lignocellulose is a complex substrate composed of a mixture ofcarbohydrate polymers (namely cellulose and hemicellulose) and lignin.The conversion of lignocellulosic biomass into ethanol relies mainly onthe efficient separation of these cell wall components to allow thehydrolysis of the carbohydrates polymer into fermentable sugars. Most ofthe processes using high temperature or pressure with acid, caustic ororganic solvent are able to provide a cellulose substrate that can bechemically or enzymatically converted into fermentable glucose (Wyman etal. (2005) Bioresource Technology 96:2026-2032; Mosier et al. (2005)Bioresource Technology 96:673-86). In general, the yield and hydrolysisrate of cellulose increases when biomass is fractionated underconditions of high temperature and extremes of pH. Under these severeconditions, however, the overall carbohydrate recovery is oftencompromised due to extensive degradation of the hemicellulose sugars(mainly xylose in hardwood), which comprise a significant fraction ofthe lignocellulosic feedstock (hardwood: Rughani and McGinnis (1989)Biotechnol. Bioeng. 33:681-686; Bakker et al. “Biofuel production fromacid-impregnated willow and switchgrass”; 2nd World Conference onBiomass for Energy, Industry and Climate Protection, 10-14 May 2004,Rome, Italy; Li et al. (2005) Appl. Biochem. Biotechnol. 125:175-88;Sassner et al. (2005) Appl. Biochem. Biotechnol. 121-124:1101-17; Pan etal. (2005) Biotechnol. Bioeng. 90:473-81; softwood: Boussaid et al.(1999) Biotechnol. Bioeng. 64:284-9; Yang and Wyman (2004) BioresourceTechnol. 86:88-95; Knauf and Moniruzzaman (2004) Intl. Sugar J.106:147-50; Mosier et al. (2005) Ibid). Also, the degradation productsgenerated by extensive hydrolysis (phenol, furans and carboxylic acid)can potentially inhibit further fermentation steps (Palmquist et al.(1999) Biotechnol. Bioeng. 63(1):46-55; Klinke et al. (2004) Appl.Microbiol. Biotechnol. 66:10-26). Furthermore, severe pre-treatmentconditions, including the use of acid catalysts, can chemically alterthe nature of the recovered lignin. A consequence of this is a decreasein the suitability of the lignin for some high value applications(Lignin Institute Dialogue Newsletter (2001) 9(1); Lora and Glasser(2002) Ibid; Matsushita and Yasuda (2003) J. Wood Sci. 49:166-171).

When water is used as the sole fractionation agent, the majority of thehemicellulose sugars can be recovered through autohydrolysis (Garroteand Parajo (2002) Wood Science Technol. 36:111-123). However, due toinefficient delignification, this maximization of the hemicellulosesugar yield is usually done at the expense of the cellulose/glucoseenzymatic conversion (Negro et al. (2003) Appl. Biochem. Biotechnol.105:87-100; Chung et al. (2005) Appl. Biochem. Biotechnol. 121:947-961;Kim and Lee (2006) Bioresource Biotechnol. 97:224-232). Use of a secondstage oxidative treatment was shown to improve the cellulose/sucroseconversion following the hot water treatment but not always as a resultof efficient lignin removal (Brownell and Saddler (1987) Biotechnol.Bioeng. 29:228-35; Wyman et al. (2005) Bioresource Technol.96:1959-1966; Kim and Holtzapple (2006) Bioresource Technol.97:583-591).

The efficient removal of lignin under mild conditions can be achievedusing the OrganoSolv™ process. This type of pre-treatment involves theuse of an aqueous organic solvent, usually ethanol, to achieve thesimultaneous removal of the hemicellulose sugar and lignin in separatedstreams. The cost associated with the use of an ethanol solvent isreduced by producing the ethanol on site and efficiently recycling it,as taught, for example, by U.S. Pat. No. 5,788,812. The conversion rateof the cellulose solid fraction provided by aqueous ethanolpre-treatment is mainly affected by the inefficient removal of thehemicellulose sugar when lower water/solvent ratios are used to maximizethe lignin recovery (Holtzapple and Humphrey (1984) Biotechnol. Bioeng.26:670-676; Chum et al., (1988) Biotechnol. Bioeng. 31:643-649).Increasing the water/ethanol ratio, or the addition of a chemicalcatalyst to the solvent, increases the hemicellulose sugar removal butis associated with a reduction of lignin removal and increasedhemicellulose sugar degradation (Holtzapple and Humphrey (1984) Ibid;Rughani and McGinnis (1989) Ibid; Pan et al. (2005) Ibid).

Successful advancements in enzyme production technology have resulted ina lower cost of the hydrolytic enzyme required to obtain a highconversion rate of cellulose to glucose. However, because the enzymatichydrolysis activity is strongly inhibited by the hydrolysis products(sucrose and short cellulose chains), simultaneous fermentation of thereleased sugar (SSF for simultaneous saccharification and fermentation)can greatly improve the overall cellulose/ethanol conversion using lowerenzyme loading. Several technologies are now available that allow abroader use of the biomass at lower cost under a variety of lessconstraining conditions (reviewed in Lin and Tanaka (2006) Appl.Microbiol. Biotechnol. 69:627-642).

Whereas the fermentation of glucose can be carried out efficiently by avariety of organisms, the bioconversion of the pentose fraction (xyloseand arabinose) presents a challenge. A lot of attention has thereforebeen focused on genetically engineering strains that can efficientlyutilize pentose and convert them to useful compounds, such as ethanol(reviewed in: Aristidou and Penttila (2000) Curr. Opin. Biotechnol.11:187-198). Alternatively, the pentose fraction which is predominantlyxylose in hardwood species such as Salix, can be recovered from thewater stream and converted to xylitol for use as a valuable food productadditive. By-product streams from this process (furfural, acetic acid,para-hydroxybenzoic acid and vanillin) may also be fractionated subjectto market price. Furfural, the easiest by-product to market, can beobtained by distillation from the same fraction. The acetic acid mayalso be recovered to produce peroxyacetic acid for pulp.

Ethanol-soluble lignin is considered to be of higher value because ofits ease of recovery and its suitability for a wide range of industrialapplications compared with water-soluble lignin, such as that recoveredfrom the Kraft process often employed by the pulp and paper industry.Extraction of Kraft lignin requires high volumes of solvent and has anarrower range of applications (Funaoka et al. (1995) Biotechnol.Bioeng. 46:545-552; Lora and Glasser (2002) J. Polymers Environ.10:39-47; Kubo and Kadla (2004) Macromol. 37:6904-6911; Lawoko et al.(2005) Biomacromol. 6:3467-3473).

Lignin extracted using the OrganoSolv™ process differs significantlyfrom that extracted via the Kraft process. OrganoSolv™ lignin has amolecular weight of 700 to 1550 g/mol, low polydispersity, a glasstransition temperature of 70 to 170° C., a high relative amount ofphenolic hydroxyl groups, and a low degree of chemical modification(Lora and Glasser (2002) Ibid; Kubo and Kadla (2004) Ibid; Lawoko et al.(2005) Ibid). This lignin can be used in the manufacture of moldingcompounds, urethane epoxy and formaldehyde resins, antioxidants andcontrolled-release agents. Ethanol-soluble lignin from hardwoods isrecovered by diluting the aqueous ethanol pre-treatment effluent withwater and acid to form a solution with a pH of 1.5 to 2.7 and an alcoholcontent of 30% (v/v) (or a ratio of aqueous-ethanol effluent to the acidwater of 0.35 to 0.70). After drying, the precipitated lignin isobtained in the form of a powder (U.S. Pat. No. 5,788,812).

Acid catalyzed OrganoSolv™ pulping was originally developed by TheodorKleinert as an environmentally preferred alternative to Kraft pulping(U.S. Pat. No. 3,585,104). It was later found that a variation of theoperating conditions could very efficiently convert the lignocellulosicmaterial to sugars and lignin. In the 1980s, a 16 liter continuous flowreactor pilot plant that processed bagasse to sugars was built (Dedini,Brazil). A concentrated solution of acetone with a small amount of acidwas used to solubilize the lignocellulosic component of the bagasse(U.S. Pat. No. 4,409,032).

An OrganoSolv™ process using aqueous ethanol to produce a clean biofuelfor turbine generators was developed by the University of Pennsylvaniaand the General Electric Company in the 1970s. Subsequent modificationby the Canadian pulp and paper industry resulted in the Alcell™ pulpingprocess (U.S. Pat. No. 4,100,016). The long-term economic viability ofthe Alcell™ process was significantly improved using technology for therecovery of lignin and furfural by-products from the organic pulpingliquor (U.S. Pat. Nos. 4,764,596, 5,681,427 and 5,788,812). A commercialAlcell™ pulping plant processing 30 metric tons of hardwood per day wasestablished in 1989 in New Brunswick Canada. The plant was operated forseveral years but was eventually shut down due to external economicfactors. More recently, a patent application was published relating toan integrated operation for processing sugarcane that combines theOrganoSolv™ Alcell™ process, pulping and fermentation to reduce thecapital and operating cost by providing a high degree of internalprocess recycling (US Patent Publication No. US 2002/0069987).

There remains a need in the art for a process for producing ethanol fromwoody biomass which can be established at a relatively low cost and beprofitable by maximizing the yield and recovery of valuable by productssuch as natural lignin and xylose.

SUMMARY

The present invention provides an integrated process that allows forrapid production of high volumes of biomass, and the efficient andcost-effective use of plant biomass for production of ethanol, naturallignin, xylose and other co-products. The process employs an optimizedpre-treatment that allows efficient fractionation of lignin andhemicelluloses without compromising ethanol yield. Due to the high costassociated with biomass production, the optimum utilization of alllignocellulosic components of the feedstock as marketable products isessential in order to obtain ethanol at a commercially competitiveprice. Due to the complex nature of the lignocellulosic components andthe technical difficulties associated with their separation andconversion, a compromise in the recovery of all valuable components isrequired to reduce the cost of producing sugars from woody biomass.

In one embodiment, the pre-treatment process, which is based on acombination of an OrganoSolv™, or ethanol/water (for example 50% to 80%ethanol in water), treatment and a hot water wash, is optimized for thefractionation of Salix, and improves the overall biomass utilization bymaximizing the lignin recovery, as well as the overall carbohydraterecovery, without compromising cellulose/glucose conversion. Productrecovery under mild conditions is further improved by applying thepre-treatment in a continuous manner (Nagle et al. (2002) Biotechnol.Prog. 18:734-738; Yang and Wyman (2004) Ibid; Wyman et al. (2005) Ibid;Liu and Wyman (2005) Bioresour. Technol. 96:1978-1985). In addition toimproving the yield of each product stream, the economy of the inventivepre-treatment process is improved by avoiding the addition of chemicalcatalysts which are expensive, require neutralization of solidfractions, chemically modify and reduce the value of the recoverablelignin, and are costly to remove from the liquid stream.

In one embodiment, the inventive process employs hardwood, preferablySalix spp, although other plant materials, such as wheat straw, may alsobe effectively processed using the methods disclosed herein. As abiomass feedstock, Salix spp. offer the advantages of requiring lowenergy input in relation to the biomass produced, being easy topropagate from unrooted cuttings, having genetic diversity and a shortbreeding cycle, having good winter standing, being inexpensive toharvest and chip, and vigorously re-sprouting after each harvest. Inaddition, growing of Salix requires significantly fewer pesticides thantraditional agriculture, uses fewer herbicides than many crops and maybe grown for ecosystem restoration (Kuzovkina and Quigley (2005) Water,Air, and Soil Pollution 162:183-204). Because Salix can grow with easeon marginal land, it is particularly suitable for restoration of usedpastoral farming land (Wilkinson (1999) Biomass Bioenergy 16:263-275).Salix culture can contribute to the reduction of nutrient leaching, soilacidification and erosion, and has been shown to improve the nitrogenbalance and increase soil fertility (Hasselgren (1998) Biomass Bioenergy15:71-74; Borjesson (1999) Biomass Bioenergy 16:137-154; Roygard et al.(2001) J. Environ. Qual. 29:1419-1432). Furthermore, like most woodycrops, Salix production is carbon dioxide neutral, and is thereforestrategically important in efforts to reduce global warming (Lemus andLal (2005) Crit. Rev. Plant Sci. 24:1-21).

The process employs a low boiling solvent, preferably ethanol, for easylignin recovery and solvent recycling. Ethanol also offers the advantagethat it is a product of the processing of cellulose and therefore can bereadily recycled as part of the biorefining process. Ethanolpretreatment without the use of an acid catalyst is preferred to achievehigh recovery of chemically unmodified natural lignin with higherpotential revenues and also to increase the recovery and integrity ofthe xylan polymer in the subsequent hot water treatment. The disclosedprocess, which in certain embodiments uses continuous processing, alsoreduces the recondensation of lignin often seen in a batch reactor byallowing removal of solvent while still at temperatures well above thenormal boiling point of the solvent.

In one embodiment, the process disclosed herein includes the followingsteps:

-   -   (a) contacting a continuous flow of plant material, such as wood        chips, with a co-current or counter-current continuous flow of        an aqueous ethanol solution (preferably comprising 50% to 80%        ethanol) at elevated temperature and pressure (for example at a        temperature between 170° C. and 210° C. and a pressure between        19-30 barg) for a period of time sufficient to produce        ethanol-soluble lignin and plant pulp material, wherein the        plant pulp material is depleted of lignin and has a high        concentration of cellulose;    -   (b) separating ethanol from the plant pulp material and        recovering the ethanol-soluble lignin from the ethanol;    -   (c) contacting the plant pulp material with water at elevated        temperature and pressure (for example at a temperature between        160° C. and 220° C. and a pressure between 12-25 barg) for a        period of time sufficient to remove hemicellulose sugars from        the plant pulp material;    -   (d) separating the water from the plant pulp material and        recovering water-soluble sugars, acetic acid and/or furfural        from the water; and.    -   (e) contacting the resulting plant pulp material with: (i) an        aqueous solution comprising cellulase, β-glucosidase and        temperature-tolerant yeast, (ii) yeast growth media, and (iii)        buffer, whereby cellulose present in the plant pulp material is        hydrolyzed to glucose.

The resulting glucose may then be fermented to produce ethanol, which isin turn recovered by way of distillation and dewatered by technologiessuch as use of a molecular sieve.

The ethanol pretreatment (step (a)) may be carried out substantially inthe absence of an acid catalyst. For example, the reaction mixture maycontain less than 1% of an acid catalyst. In certain embodiments, theethanol pretreatment is carried out at a pH in the range of 3 to 9.5.Similarly, in certain embodiments, the hot water treatment (step (c)) iscarried out at a pH in the range of 2 to 7.

Methods for propagating plants of a Salix species are also providedherein. In certain embodiments, such methods comprise:

-   -   (a) culturing at least one shoot of a first plant selected from        the group consisting of Salix species and collecting at least        one cutting from the shoot, wherein the cutting contains at        least one node;    -   (b) cultivating the cutting in a composition for a period of        time sufficient to form a second plant, wherein the composition        comprises benzyladenine, activated charcoal and at least one        medium selected from the group consisting of: Murashige and        Skoog medium and McCown Woody Plant medium;    -   (c) obtaining at least one subsequent cutting from the second        plant, wherein the subsequent cutting contains at least one        node; and    -   (d) culturing the subsequent cutting in McCown Woody Plant        medium for a period of time sufficient to form a subsequent        plantlet.

Plants that may be effectively propagated using such methods include,but are not limited to, S. viminalis and S. schwerinii ‘Kinuyanagi’.

These and additional features of the present invention and the manner ofobtaining them will become apparent, and the invention will be bestunderstood, by reference to the following more detailed description andthe accompanying drawings.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a schematic of the first stage (ethanol extraction) of thedisclosed integrated process for the production of biofuel from woodchips.

FIG. 2 is a schematic of the second stage (hot water treatment) of thedisclosed integrated process for the production of biofuel from woodchips.

FIG. 3 is a schematic of the third stage (simultaneous saccharificationand fermentation) of the disclosed integrated process for the productionof biofuel from wood chips.

FIG. 4 is a schematic of the fourth stage (productseparation/purification) of the disclosed integrated process for theproduction of biofuel from wood chips.

FIG. 5 is a schematic of an experimental 100 ml digestor for thepre-treatment of biomass

FIG. 6 shows a schematic of a 3 l packed-bed experimental digestor forthe pre-treatment of biomass

FIG. 7 is a schematic of a 40 l batch experimental digestor for thepre-treatment of biomass.

FIG. 8 is a graph showing the effect of time on the DM (dry mass)removed from Salix chips with 70% ethanol solvent at three differentscales (100 ml, 3 l and 40 l), expressed as a percentage of the initialDM loaded.

FIGS. 9A and B shows the ratio of DM and lignin removed from Salix chipswith 70% ethanol and expressed as a percentage of the initial DM loaded.FIG. 9A shows the results obtained with the 3 l digestor, and FIG. 9Bshows the results from individual experiments in the 40 l batchdigestor.

FIG. 10 is a graph showing the effect of time on the DM removed fromSalix chips with water solvent applied as a primary treatment or afteran ethanol treatment at two different scales (100 ml, 3 l), expressed asa percentage of the initial DM loaded.

FIG. 11 is a graph showing the proportion of lignin in the DM removedfrom Salix chips with 70% ethanol in three different scales (100 ml, 3 land 40 l), expressed as a percentage of the total DM removed.

FIG. 12 is a graph showing the proportion of the total available ligninrecovered after pre-treatment of the Salix chips with 70% ethanol in the100 ml, 3 l and 40 l digestors, expressed as a percentage of the lignincontent in the untreated chips.

FIG. 13 shows the ratio of DM and lignin that was removed from Salixchips with hot waters applied as a primary treatment or, after a 70%ethanol treatment in the 100 ml and 3 l digestors, expressed as apercentage of the initial DM loaded.

FIG. 14 is a graph showing the conversion of cellulose recoveredfollowing Organosolv™/liquid hot water treatment of Salix chips intoglucose by enzymatic hydrolysis, compared with commercially obtainedpure cellulose and cellulose recovery from untreated Salix chips.

FIG. 15 is a graph showing the accumulation of xylose, furfural, aceticacid and glucose during the hot water treatment of 70% ethanol treatedSalix chips in the 3 l digestor.

DETAILED DESCRIPTION

As discussed above, the present invention provides aneconomically-viable integrated process for the biorefining oflignocellulosic material from plants, such as Salix spp., to produceethanol and natural lignin. Other types of feedstock that may beeffectively employed in the disclosed process include dedicated shortrotation woody or herbaceous biomass (for example, Miscanthus,switchgrass), woody and agricultural waste (e.g., wheat straw, ricestraw, corn stover or sugar cane bagasse) and dedicated energy crops. Incertain embodiments, the plant material is selected from the groupconsisting of: Salix, Poplar, Eucalyptus, switch grass, miscanthus,sugar cane bagasse, soybean stover, corn stover, rice straw, barleystraw, wheat straw, corn fiber, wood fiber, and combinations thereof.

As used herein, the term “woody plant” refers to a vascular plant thathas at least one stem that is lignified to a high degree. Examples ofwoody plants include trees and shrubs. Salix crops may be grown frommicropropagated plants as described below in Example 3. Salix speciesthat may be effectively employed in the inventive process includegenetically modified species.

In one embodiment, the process employs a continuous flow counter-currentor co-current digestor. Use of such a digestor results in fasterprocessing rates, increased throughput and increased efficiency. As suchdigestors run continuously, they require less maintenance and less laborthan batch digestors. In addition, displacement wood pulping is moreefficient than batch processes and differential reaction times arepossible.

Crops of Salix are harvested, air-dried and stockpiled. If reduction ofthe particle size of the harvested Salix is desired prior to processing,this can be achieved using a chipper or similar device. In oneembodiment, Salix particles of approximately 5 mm to 5 cm in size areemployed in the process disclosed herein.

The first stage of the process disclosed herein is an OrganoSolv™, orethanol, extraction (illustrated schematically in FIG. 1). This involvescontinuous contacting of the wood chips with a counter-current flow of asolution of up to 70% ethanol in water, undertaken at a temperature ofapproximately 170° C. to 210° C. and a pressure of 19-30 barg. In oneembodiment, the digestor is a screw contactor operating with wood chipsbeing fed and discharged via cup and cone pressure plugs or feed screws.Solvent passes against the flow of solids so that chips exiting themachine are exposed to fresh (solute free) ethanol solution, while chipsentering the digestor, which have the highest extractable content, areexposed to the most solute laden solvent solution. Solvent entering thedigestor is pressure pumped to maintain the operating pressure thereinand to provide the hydraulic drive to pass against the flow of chips.Solvent from within the digestor is re-circulated through externalheaters, for example steam heaters, on a continuous basis to bring thewood chips up to the operating temperature quickly and to maintain thetemperature. Operating conditions (such as time, temperature profile,pressure and solid/liquid ratio) within the digestor are optimized toprovide maximum removal of water insoluble lignin from the wood chips.As the wood chips pass from the digestor and are exposed to lowerpressures, a portion of the solvent content therein will evaporate,resulting in cooling of the wood chips.

In an alternative embodiment, the wood chips are displaced in thedigestor using gravity in a downward gradient. Solvent entering thedigestor is pumped upward passing against the flow of solid.

Chips discharged from this first stage of the process will still containsome ethanol which must be removed prior to the subsequent waterextraction. This is achieved by means of a steam stripping operation.The vapors are recovered from both this operation and from the flashevaporation of depressurized solids, noted above, and are re-useddirectly with the fresh solvent stream. In this way the latent heatcontent of the vapors is recovered.

The de-solventized and lignin-depleted chips then pass into a secondstage of extraction (illustrated schematically in FIG. 2) undertaken incomparable equipment, and in a comparable fashion to the ethanolextraction described above, with the difference being that high pressurehot water (preferably at a pressure of approximately 12 to 25 barg and atemperature of approximately 180° C. to 220° C.) is utilized tosolubilize the xylose fraction of the chips.

As the solids exit the hot water digestor and the pressure is reduced,flash evaporation of steam will occur. This is recovered for directre-use with the fresh hot water entering as fresh extraction solvent atthe solids discharge end of the digestor. The chips will be cooled as aresult of this flash evaporation.

What remains of the initial wood chips after two stages of extractionwill be primarily cellulose in a hydrolyzable pulp. This material istransferred to one of a series of batch SSF (simultaneoussaccharification and fermentation) vessels, together withtemperature-tolerant yeast, yeast growth media, cellulase,β-glucosidase, buffer and water to dilute the solids to the requiredsolid/liquid ratio (illustrated schematically in FIG. 3). In thesevessels, the cellulose is hydrolyzed to produce glucose, which is inturn fermented to produce ethanol. Low levels of ethanol are maintainedin the fermentor by continuous removal of the produced ethanol to avoidfermentation inhibition. The process is optimized for maximum cellulosehydrolysis and fermentation to ethanol. The vessel contents at the endof the batch fermentation will be discharged via a filter and theretained solids will be recovered for disposal as cattle feed. Residualcomponents in this fraction may also be recovered.

The filtrate, consisting primarily of ethanol and water, is concentratedto produce hydrous and/or anhydrous ethanol as desired, using methodswell known to those of skill in the art. A portion of the hydrousethanol product is re-utilized in the first, ethanol extraction, stage.

Additional products are separated and purified as illustratedschematically in FIG. 4 and discussed in detail below.

Lignin Recovery

The black liquor (ethanol/water/lignin solution) exiting the ethanoldigester in the first stage is depressurized before passing to a flashcooling vessel in which the solvent (primarily ethanol) is evaporated.Further ethanol is then steam-stripped from the liquor prior to transferto one of a series of batch vessels in which precipitation of ligninfrom the liquor is promoted through dilution (3 to 10 times) with waterand lowering of pH (<3) by acid addition. The resulting ligninprecipitate is then separated by filtration and dried as a crude productstream. The aqueous filtrate is combined with the hot water stream forxylose and water soluble product recovery.

Xylose Recovery

The hot water extraction product stream from the second stage containingprimarily xylose (with some low molecular weight lignin, some glucose,and other C5 and C6 sugars) is depressurized before cooling by flashevaporation of water. As the temperature is dropped, the low molecularweight compounds and molecules precipitate from solution. These are thenseparated by filtration.

The filtrate from the low molecular weight filtration contains thexylose fraction as well as a range of other components includingfurfural, acetic acid, para-hydroxybenzoic acid and vanillin. Anadditional module carries out concentration, decolorization,deionization and chromatography steps, and produces pure xylose.

Solvent Recycling

The ethanol and water streams can be recycled through the pulp biomassto increase product concentration, or processed for product recovery.Subject to processing conditions during the two extraction operations,varying degrees of at least acetic acid and/or furfural will becontained in the stream passing to the ethanol concentrator. Thesefractions from the ethanol/water distillation can be concentrated andrecovered using methods well known in the art.

The following examples are offered by way of illustration and not by wayof limitation.

EXAMPLE 1 Biorefining of Salix Biomass

Preparation and Composition Analysis of Untreated Salix Biomass

Stems of Salix viminalis or Salix schwerinii ‘Kinuyanagi’ were chippedwith a garden mulcher. The wood chips were dried at 40° C. for 24 hoursand sieved by hand between two wire meshes of British test sieve withapertures of 2.8 and 4 mm. The composition of the sieved and unsievedSalix chips was assessed, with the results being shown in Table 1. Themass composition was assessed using laboratory analytical procedures(LAPs) developed by the National Renewable Energy Laboratory (NREL,Golden, Colo.). Values are expressed as gram of component per 100 g ofdry untreated chips. Extractives were isolated using a Soxhletextractor, dried and weighed. Lignin concentrations were determinedafter chemical hydrolysis of the Salix chips (4 hours with 72% sulfuricacid at 102° C.). Acid soluble lignin was measured by densitometry at320 nm and the concentration of the non-acid soluble lignin was measuredby weight minus ash. The percentage of glucan and xylan present in thesamples were determined after chemical hydrolysis (4 hours with 72%sulfuric acid at 102° C.). Acid soluble sugar was measured by HPLC usingthe appropriate range of xylose and glucose standards.

TABLE 1 Composition of untreated Salix biomass Extractive Lignin (%)Sugar (%) Salix variety (%) Soluble Insoluble Total Glucan Xylan Salixviminalis* 16 2 31 33 23 9 Salix viminalis 8 3 24 27 34 8 Salixschwerinii 6 5 23 28 32 14 Salix schwerinii 4 5 22 27 33 12 KinuyanagiSalix schwerinii 4 3 25 28 33 9 Kinuyanagi Salix schwerinii 2 4 28 32 359 Kinuyanagi + Salix viminalis Salix schwerinii 2 4 25 29 30 8Kinuyanagi + Salix viminalis Average 6 4 25 29 31 10 Standard 5 1 3 3 42 Deviation (*= Sieved material)Pre-Treatment of Salix Biomass

The pre-treatment of Salix chips was tested in 100 ml experimentaldigestor and 3 l packed-bed experimental digestor that were able toprocess 6 g and 300 g of dry wood chips, respectively. The design ofthese two digestors is illustrated in FIG. 5 (100 ml digestor) and FIG.6 (3 l packed-bed digestor). A 40 l digestor was also designed andtested for the recovery of natural lignin from Salix biomass at largerscale (FIG. 7). This 40 l digestor is able to process 6 kg of drybiomass.

Description of the 100 ml Experimental Digestor (FIG. 5)

The 100 ml capacity experimental pre-treatment digestor 1 consisted of aone inch tube 10 with an externally coiled heating coil 11 andfiberglass insulation 19. Tube 10 was connected to a Swagelok™ end-cap12 which in turn was connected to a welded pressure transducer 13 bymeans of a ¼ inch tube 14 and to a thermocouple port 15. The other endof tube 10 was connected to a one inch integral bonnet needle valve 16connected to a collection tube 17 of the same length, which in turn wasconnected to a ¼ inch integral bonnet needle valve 20. The temperaturewas controlled by a thermocouple 18 wedged underneath the heating coiland connected to a controller 21. This configuration facilitated theremoval of solvent at a temperature well above the boiling point of thesolvent.

Operation of the 100 ml Experimental Pre-Treatment Digestor

For the ethanol pre-treatment, the digestor of FIG. 5 was loaded with6.54 g of dried wood chips and 83.5 ml of ethanol (50 to 70%). Thedigestor was sealed at all Swagelok™ fittings and the bonnet needlevalve closed. The digestor was then heated to the selected processtemperature (170° C.-195° C.) while being agitated manually to ensurethat the process reached equilibrium quickly. Once the desiredtemperature was reached, the reaction was allowed to proceed for 60minutes with periodic manual agitation. At the end of the reaction time,the digestor was inverted and the bonnet needle valve opened to allowthe solvent to drain into the collection tube. A fine mesh, positionedin the digestor against the valve, retained the solid fraction in thereaction vessel. The content of the digestor was cooled down and thesolvent was removed from the collection tube.

For the hot water treatment, the digestor was filled with 90 ml of washwater, sealed and heated to a specified temperature in the range of 180to 220° C. After the desired incubation time at the target temperature,the hot water was removed using the same method as described for solventremoval. The remaining pulp was dried and submitted to hydrolysis.

Description of the 3 l Packed-Bed Digestor (FIG. 6)

The 3 l packed-bed digestor 23 shown in FIG. 6 consisted of a stainlesssteel digestion chamber 25 housing a wire mesh sample basket 27 andsealed with Swagelok™ end cap fittings 26. There were four outlets andtwo inlets from the digestion chamber 25. A ¼″ tube outlet 24 connectedthe digestion chamber 25 to a pressure transducer 32. A ½″ tube pressurerelief valve outlet 28 housed a pressure thermocouple 39 measuring thepressure in digestion chamber 25, and was connected to a 50 bargpressure relief valve 38 and a water tank inlet 54. A ¾″ tubecirculating fluid inlet 33 allowed re-circulation of fluids intodigestion chamber 25 and application of pressure from a pressurizednitrogen cylinder through a nitrogen cylinder connector 44. A 1″ tubecirculating fluid outlet 40 allowed re-circulation of circulating fluidsout of digestion chamber 25 and a ⅛″ tube thermocouple inlet 55 measuredthe temperature inside the digestion chamber 25. A thermocouple 56connected to a circulating fluid tube 45 measured the temperature of thecirculating fluid in the circulating fluid tube.

A motor 34 rotated a shaft 46 housed in a 2″ tube 48 that was connectedto a variable speed drive pump 31 containing four propellers 47 andsealed using several ECOFLON₂ rotary seals. Pump shaft T pieces 49 heldthe 2″ tube 48 in place. When shaft 46 is rotated at a speed of1,400-2,800 rpm, fluid is forced through pump 31 and circulated throughheating loop 29 containing a heater construction 35, and through thedigestion chamber 25 to enable co- or counter-current continuous flow.

A recycle line T piece 51 was connected to a needle valve 36 and a 10 mlbottle 37 to enable taking of circulating fluid samples when thedigestor is operating. To take a sample of circulating fluid, valve 36was opened and bottle 37 filled. The valve 36 was then closed, andbottle 37 was cooled and removed for sample analysis.

The heater construction 35 consisted of a ¾″ heating tube 52 with sixelectrical heating elements 53 sealed onto it with conductive cement.The heating elements 53 were connected to a controller 41, which wasconnected to a control thermocouple 42 measuring the temperature of thecirculating fluid in the middle of heating loop 29, and anover-temperature controller 43 set at 250° C. and measuring thetemperature of the circulating fluid near an outlet 50 of heating loop29. The heating tube 52 was insulated with fiberglass.

Operation of the 3 l Packed-Bed Digestor

The 3 l packed-bed digestor shown in FIG. 6 functioned under the sameprinciple as the 100 ml digestor with the exception that the digestionchamber 25 contained the biomass within wire mesh sample basket 27 andthe solvent was circulated within the heating loop 29 by way of variablespeed drive pump 31. The solvent was heated electrically to ensure thatthe target temperature for digestion was reached. The kinetics of theextraction process was determined by collecting samples of the mobilesolvent by way of needle valve 36 and 10 ml bottle 37 situateddownstream of the pump.

The chipped wood feedstock (up to 300 g) was placed in the wire meshsample basket 27, which fitted tightly inside the vessel. The vesselwith the sample basket was filled with up to 3 l of solvent, and thereactor was sealed tightly with Swagelok™ fittings. The recycle loop wasfilled with liquid by adding water through the water tank inlet 54. Whenthe reactor was sealed completely, the circulator and temperaturecontrollers were switched on. The pressure transducer 32, thermocouples39 and 55 in the reactor, and thermocouple 56 on the tube surface 45were monitored using a PicoLog Recorder (Pico Technology, Cambridge,UK).

Description and Operation of the 40 l Batch Digestor (FIG. 7)

The 40 l packed-bed digestor shown in FIG. 7 was fabricated from asection of 210 mm high-pressure mild steel tubing which formed a reactorvessel 64. A surrounding 300 mm tube formed a heating coil 63 which waspartitioned by a spiral steel baffle into a spiral flow channel. Thereactor vessel 64 and heating coil 63 were surrounded by glass fiberinsulation 62. The heating coil 63 was connected to an oil heatingcircuit 76 and oil inside the oil heating circuit 76 was heated by aheater 69 driven by 6 kW of electrical heating elements. The oil wascirculated through the oil heating circuit 76 by a pump 68, and couldalso be diverted by two loop valves 71 to a cooling loop 72 immersed ina water bath 77 to enable faster cooling of the reactor vessel 64. Theheater and pump were controlled by a thermostat 70 and a processcontroller 67. An oil reservoir 73 connected to the oil heating circuit76 was used to accommodate thermal expansion of the oil. In operation,the reactor vessel 64 was filled with about 6 kg of Salix biomass and 30to 40 liters of 70% ethanol via a funnel 57 and an upper ball valve 58.The reactor vessel 64 was then sealed and heated, taking 3 to 4 hours toreach operating temperature (185° C.). The temperature was monitored bya temperature probe 61 and the pressure by a pressure gauge 59. Thepressure gauge was connected to a pressure relief valve 60. A samplingport 66 was attached to a lower ball valve 65 in the reactor vessel 64.To enable time-course samples of the liquid phase to be taken during theextraction step, a liquid sampling valve (not shown) can be attached tothe sampling port 66. After the appropriate residence time, oilcirculation was switched to the cooling loop 72 by means of the loopvalves 71 and the reactor vessel 64 allowed to cool. After removal ofthe liquid sampling valve 74, the treated biomass in the form oflignin-containing black liquor was drained from the reactor vessel 64 bymeans of the lower ball valve 65 and sampling port 66, and washed with70% ethanol and water to remove additional lignin. Alternatively,attachment of a steam explosion valve (not shown) to the sampling port66 enabled a steam explosion step to be performed on the biomass whilestill at high temperature and pressure.

Results

Mass Balance

Using the experimental digestors as described above, Salix biomass wasfractionated into two fractions: 1) an ethanol and/or water solublefraction (hydrolysate, Hyd.), and 2) a solid fraction (pulp). Table 2represents the mass partition of the Salix chips following variouspre-treatment sequences. Treatments were done with 70% ethanol at 170°C. to 190° C. for 60 minutes either before or after water treatmentsperformed for 30 min at 170° C. to 190° C. In this example, allpre-treatment experiments were initiated with 6.54 g of dry Salix chips(n=3-5) in the 100 ml digestor. The mass in the hydrolysate representsthe dry mass (DM) recovered after evaporation of hydrolysate, and themass in the pulp fraction corresponded to the DM of the residualinsoluble material yielded after each pre-treatment. These results showthat the addition of a second pre-treatment increased the displacementof mass by 10% toward the hydrolysate and that the sequence in which thetwo treatments are performed does not have a great impact on the finalamount of mass displaced.

TABLE 2 Mass balance of pre-treated Salix biomass Mass in hydrolysateMass in pulp fraction Treatment g ± Std Dev % ± Std Dev g ± Std Dev % ±Std Dev Organosolv ™ 1.87 ± 0.26 28.53 ± 4.04 4.79 ± 0.06 73.24 ± 0.92Hot water 1.72 ± 0.19 26.24 ± 2.91 4.53 ± 0.07 69.27 ± 1.07Organosolv ™- 2.91 ± 0.32 44.40 ± 5.00 3.81 ± 0.05 58.26 ± 1.68 Hotwater Hot water- 2.75 ± 0.26 41.96 ± 3.93 3.72 ± 0.05 56.88 ± 1.83Organosolv ™

The kinetics of mass removal during the primary treatment with 70%ethanol was studied in the 3 l packed-bed digestor. FIG. 10 presents theaverage amount of DM removed (expressed as a percentage of the initialDM loaded) in a set of five experiments performed in the 3 l packed-beddigestor with 70% ethanol for 60 to 480 minutes at a temperature varyingbetween 175° C. and 195° C. This set of data, obtained from thepre-treatment of 250 g of dry Salix chips, was compared with thepercentage of mass removed with 70% ethanol using the smaller (100 ml;6.54 g) or larger (40 l batch; 30 to 40 kg) digestors at various times.

FIG. 8 shows that a three times longer incubation was required using the3 l packed-bed digestor to remove comparable amounts of dry matter asthe smaller, 100 ml digestor in 60 min. Therefore, incubation timesvarying between 200 and 400 minutes were assayed for optimum massremoval in the larger 40 l digestor. As shown in FIG. 8, in this rangeof incubation time, 20 to 30% of the input DM was efficiently removedwith 70% ethanol at the 40 l scale. FIG. 9 illustrates the ratio of drymatter and lignin removed by the 70% ethanol as the percentage of the DMloaded, with the results for the 3 l digestor being shown in FIG. 9A andthe results for the 40 l batch digestor being shown in FIG. 9B. Theaverage DM removed in 22 extractions with 70% ethanol in the 40 l batchdigestor (FIG. 9B) was 25%±3 in a running time varying between 200 and400 minutes at an initial liquid loading ratio (iLLR; 1 solvent per kgiDM).

FIG. 10 presents the ratio of DM removed when untreated Salix dry chipsand Salix dry chips pre-treated with 70% ethanol were treated with hotwater (170° C. to 195° C.) in the 100 ml and 3 l digestors. As seenearlier with the 70% ethanol treatment, the ratio of DM remove removedwas lower when using the 3 l packed-bed as compared with the smaller 100ml digestor. FIG. 10 also shows that an increased incubation time of theuntreated chips in the hot water did not result in an increase of DMremoved as it did for longer incubation in 70% ethanol (shown in FIG.8). As in the 100 ml digestor, when the hot water treatment of Salixchips that were pre-treated with 70% ethanol was performed in the 3 lpacked-bed digestor, an additional 10% of DM removal was achieved.

Mass Composition

The Organosolv™/hot water sequence gave optimum lignin and sugarrecovery. Table 3 below shows the representative composition of thehydrolysate and pulp fraction obtained after sequential treatment of6.54 g, 250 g or 35 kg of Salix chips with 70% ethanol at 175° C. to195° C. for 60 to 345 minutes followed by water treatment at 170° C. to195° C. for 30 to 375 minutes.

The composition of the comparative untreated Salix was the average ofthe analysis of untreated Salix varieties described in Table 1. Theconcentration of lignin in the hydrolysate sample was determined afteraqueous acid precipitation of the lignin, separation and drying andweighting of the precipitate lignin. This weight measurement of ligninconcentration was shown to correlate with measurement obtained by sizeexclusion chromatography of the same precipitated lignin andinterpretation of the retention time with reference to appropriatepre-run peptide standards. The glucose and xylan concentration in thehydrolysate was directly measured by HPLC using the appropriate range ofstandards. The composition of the pulp was assessed as described earlierfor the untreated Salix chips.

Lignin Recovery

At all scales (100 ml, 3 l packed-bed, and 40 l batch), the sequential70% ethanol and hot water treatment resulted in the removal of over 30%of the total lignin content of the untreated chips (Table 3 below). Themajority of the lignin (28 to 32%) was solubilized during the primarytreatment with 70% ethanol solvent and an additional 3 to 8% of theinitial lignin was removed in the subsequent water treatment.

As shown in FIG. 11, the ratio of lignin to DM removed by the 70%ethanol treatment reached 35% in the first hour of treatment at atemperature of 170° C. to 190° C. using the 100 ml and the 3 lpacked-bed digestors. The lignin composition of the DM removed in the 3l packed-bed digestor during the second hour of treatment increased by5% and reached 50% after 4 hours. After 8 hours, the lignin content ofthe DM removed increased only by another 10% to reach 60%. FIG. 11 alsoshows that, in the 40 l batch digestor, the ratio of lignin to DMremoved varied between 30 to 48% when Salix dry chips were treated with70% ethanol solvent. FIG. 12 illustrates the proportion of the totallignin content in the untreated chips that was recovered in the 70%ethanol solvent using each of the three digestors. The higher recoveryof lignin (32%±3) in 60 minutes, using the smaller 100 ml digestor,reflects the higher rate of DM removal achieved with this digestor. Withthe 3 l packed-bed digestor, similar recovery was achieved within 200 to240 minutes of treatment. The amount of lignin recovered using the 40 lbatch digestor varied between 22 and 44% of the initial lignin contentof the Salix chips corresponding to 6 to 13% of the initially DM loaded.

FIG. 13 shows the ratio of DM and lignin removed by hot water treatmentusing the 100 ml and 3 l packed-bed digestors, expressed as a percentageof the DM loaded. When the hot water was applied as a primary treatment,up to 3% of the initial DM was rapidly recovered as lignin in the watersolvent (within the first 30 min of treatment, corresponding to 10% oftotal lignin available). When the hot water was applied after the 70%ethanol treatment, no more than 1% of the initial DM was recovered aslignin in the water solvent (3% of the total available lignin in theuntreated Salix chips).

The lignin precipitated from the ethanol hydrolysate by addition ofacidic water had an average molecular weight of approximately 2,000Daltons and was estimated to be small pentameric to decameric polymerswith a guaiacyl:syringyl unit ration of 1:4 as shown by NMR spectroscopyanalysis. NMR analysis also showed that the Salix lignin underwentlittle modification under the optimum pre-treatment conditions (70%ethanol at 195° C. for 60 minutes).

Table 3 shows the composition of treated Salix wood chips afterpre-treatment with 70% ethanol at 175° C. to 195° C. (60 min in 100 mldigestor, 180 min in the 3 l packed-bed digestor and 345 min in the 40 lbatch digestor) followed by water treatment at 170° C. to 190° C. (30min in the 100 ml digestor, 180 min in the 3 l packed-bed digestor and375 min in the 40 l batch digestor), compared with untreated Salix woodchips.

TABLE 3 Compo- sition Un- Treated Composition of solid and liquid streamProduct Recovery (% of total Scale & Salix chips after chipspretreatment (% of total DW In) component in untreated chips) Compo- (%DW Hydrolysate Hydrolysate nent chips) Ethanol Water Pulp Total EthanolWater Pulp Total 100 ml DM 26.5 ± 4.04 17.9 ± 5.00 58.3 ± 1.83 102.7Lignin 29 ± 3 9.3 1.0 10.5 21 32 3 36 72 Glucan 31 ± 4 nd nd 30.4 30 ndnd 98 98 Xylan 10 ± 2 nd 2.1 4.6 7 nd 21 46 67 Other 30 17.2 14.8 12.845 57 49 43 149 3 l DM 25 15 60 100 Lignin 29 ± 3 8.1 0.5 12.2 21 28 242 72 Glucan 31 ± 4 0.1 1.25 38.0 39 0 4 123 127 Xylan 10 ± 2 0.4 2.40.04 3 4 24 0 28 Other 30 16.38 10.9 9.8 37 55 36 33 123 40 l DM 26 1460 100 Lignin 29 ± 3 9.1 nd 17.0 26 31 nd 59 90 Glucan 31 ± 4 nd nd 32.633 nd nd 105 105 Xylan 10 ± 2 0.0 nd 0 0 0 nd 0 0 Other 30 17.0 nd 10.427 56 nd 35 91Glucose Recovery and Fermentation to Ethanol

Pre-treatment of Salix chips yielded most of the cellulose in the pulpas shown by the recovery of more than 98% of the total input glucan inthis fraction at each of the digestor scales tested (100 ml, 3 l or 40 ldigestors, Table 3). As shown in FIG. 15 and Table 4, completeconversion of the recovered cellulose into glucose was achieved after astandard five hours treatment with cellulase (Trichoderma reesei(Celluclast, Novozyme, Denmark)) at 80 pfu per gram of glucan(theoretical) and 0.05% beta glucosidase (Aspergillus niger (Novozyme188)) as recommended by the enzyme manufacturer. Also, this resultindicated that the residual lignin and xylose in the pulp, at celluloseloading ratio of 1%, did not interfere with the enzyme activity (Tables3 and 4). This provided a glucose substrate at a concentration of 10 to12 g per liter for fermentation to ethanol.

TABLE 4 Efficiency of enzymatic digestion of pre-treated Salix chips atconstant enzyme loading (80 pfu/g cellulose) Cellulose loading Pre- (%in enzyme Cellulose Digestor Treatment reaction) Agitation digestion (%)100 ml 70% ethanol 1 Shaking 100%  and hot water 1 Shaking 100%   3 l70% ethanol 5 Shaking 46.31%   4 Shaking 51.91%    40 l 70% ethanol 4Shaking 65% and hot water 5 Shaking 76% 6 Rolling 91% 8 Shaking 61% 8Rolling 70% 11 Shaking 41%

The effect of the hot water treatment on the hydrolysis of the 70%ethanol treated chips was observed when the enzymatic reaction wasperformed using higher concentration of cellulose (cellulose loadingratio>4). As shown in Table 4, over 20% more glucose was produced atequivalent cellulose loading of 4-5%, when the 70% ethanol pre-treatedchips were also treated with hot water. This improvement of cellulosedigestion at higher loading ratio probably reflected the lower contentof lignin and xylose observed in the pulp provided after hot watertreatment.

The digestibility of the cellulose yielded by the 70% ethanol and hotwater treatment was further improved by providing agitation usingrollers instead of using a flask shaker during the enzymatic reaction(Table 4).

Glucose was fermented to ethanol using 64 ml hydrolysate and 4.5 mlStill Spirits Temperature Tolerant Turbo Yeast (Brewcraft USA, Portland,Oreg.) in 6.4 ml 10× YP medium (YP medium: 100 g/l yeast extract and 200g/l peptone). The reaction was allowed to proceed at 40° C. withagitation at 200 rpm until the growth curve of the yeast had reached aplateau, determined by measuring the OD₆₀₀ of hourly samples. The amountof ethanol and remaining glucose in the medium was determined by HPLC.The yield of ethanol from the digested Salix cellulose usingSaccharomyces cerevisiae was 0.32 g of ethanol per g of glucoserepresenting 62% of the theoretical yield of 0.51 g of ethanol per g ofglucose.

Xylose Recovery

Because very small amounts of xylose and acetic acid were detected inthe 70% ethanol solvent after the pre-treatment of the Salix chips(Table 3), we concluded that 70% ethanol treatments have little effecton the hemicellulose degradation. The recovery of hemicellulose sugarsvaried according to the hot water pre-treatment conditions.

When a short hot water treatment (30 min) was performed on Salix chipspre-treated with 70% ethanol in the 100 ml digestor, residual xylose inthe pulp fraction (4.6% of DM loaded=46% xylose available; Table 3)indicated an incomplete hemicellulose break down.

FIG. 15 shows the level of accumulation of xylose and furfural in thehot water applied after the 70% ethanol pre-treatment in the 3 lpacked-bed digestor. The level of xylose peaked at 2.4% of the DM loaded(24% of the total xylose available) after 120 min, at which time thelevel of furfural production increased, indicating further degradationof the xylose yielded through efficient hydrolysis of hemicellulosesugar. As a result, the cellulose pulp that was produced after the 70%ethanol pre-treatment and longer hot water treatment contained greatlyreduced levels of xylose (Table 3, <0.1% of DM loaded). Anotherindication of efficient hemicellulose hydrolysis during the hot watertreatment was the formation of acetic acid as a consequence of thedeacetylation of the acetylated moiety of hemicellulose (FIG. 15). FIG.15 also shows that the amount of glucose was maintained at a low levelduring all times, indicating that the hot water treatment did not resultin cellulose hydrolysis.

The importance on xylose recovery of applying the hot water treatmentafter the 70% ethanol pre-treatment was further demonstrated in the 40 lbatch scale digestor. No xylose was detected in the cellulose pulpproduced after the 70% ethanol and hot water treatment whereas xyloselevels up to 7% of DM loaded (70% of total xylose) was measured in thecellulose pulp produced by the 70% ethanol treatment.

EXAMPLE 2 Biorefining of Wheat Straw

Table 5 shows the mass composition of untreated wheat straw and thecomposition of the hydrolysates and pulp produced during ethanol-waterpre-treatment of the same wheat straw. The pre-treatment was applied asdescribed above for the ethanol-water pre-treatment of the Salix chipsin the 40 l batch digestor.

As seen in Table 5, only 27% of the initial dry matter was removedduring sequential extraction with 70% ethanol and hot water. Therefore,less lignin was recovered in the ethanol solvent than when Salix chipswere treated the same way. Also, a higher proportion of lignin was foundin the wheat straw pulp. This may reflect the different type of ligninin wheat straw. As for the Salix pre-treatment, the pre-treatment ofwheat straw with ethanol and water resulted in the recovery of allavailable glucose in the pulp.

TABLE 5 Composition of solid and Composition liquid stream after chippre- Product Recovery (% of total Un-Treated treatment (% of total DWIn) component in untreated chips) Scale & Wheat Straw HydrolysateHydrolysate Component (% DW chips) Ethanol Water Pulp Ethanol Water Pulp40 l DM 19 8 72 Lignin 27 6 1 14 22 4 52 Glucan 37 nd <1 37 nd 0.3 100Xylan 19 nd <1 3 nd 1 17

When the pulp was submitted to enzymatic hydrolysis as described earlierfor the Salix ethanol-water extracted pulp (cellulose loading ratio of 5and cellulase loading of 80 fpu per gram), the cellulose in the wheatpulp was completely hydrolyzed (100%) within 6 days of reaction agitatedusing rollers.

EXAMPLE 3 Micropropagation of Salix Spp

The technique of micropropagation was used to rapidly develop largenumbers of clonal Salix spp. plantlets at low cost. Planting stakes ofS. viminalis and S. schwerinii ‘Kinuyanagi’ were produced viamicropropagation as follows.

To establish shoot cultures in vitro, stems from one-year-old Salixspecies grown in the field were collected in winter and cut into 25 to35 cm long cuttings. The cuttings were washed in water, sterilized in15% commercial bleach for 15 min and rinsed three times in water. Thecuttings were then placed in a beaker containing water. Four to sixweeks later, new shoots (5 to 10 cm long) were produced from thecuttings. The new shoots were collected and sterilized in 15% bleachafter leaves were removed. The sterilized shoots were rinsed three timesin sterile water in a sterile tissue culture hood. The shoots were thencut into 0.5 to 1 cm long micro-cuttings containing two nodes each. Themicro-cuttings were placed into MS (Murashige and Skoog) medium (Sigma,St Louis Mo.; Murashige and Skoog, Physiol. Plant. 15:473-497, 1962) orM^(c)Cown Woody Plant medium (Duchefa, Haarlem, Netherlands; Lloyd andMcCown, Proc. Int. Plant Prop. Soc. 30:421-427, 1981) supplemented with0.1 to 1.0 mg/l BA (benzyladenine) and 0.1 to 1.0 g/l activatedcharcoal, and incubated in a plant growth room at 24° C. with a 16-hourphotoperiod. Four weeks later, a shoot (2-4 cm long) and several rootswere produced from each micro-cutting to form a plantlet. The plantletswere cut again into micro-cuttings and cultured in M^(c)Cown Woody Plantmedium to increase the number of plantlets. This process may be repeatedevery four weeks. Plantlets were transplanted into potting mix in 25 mlcells.

Following transfer in soil, plantlets were kept in growth chambercontaining 100% humidity for one week before being exposed to normalhumidity conditions. Four weeks after transfer into potting mix,plantlets of 10-15 cm high were cut into 3-5 cm segments (containing aleast 2 nodes) that were re-planted in potting mix in 25 ml cells tofurther increase the number of plantlets. Alternatively, the plantletscan be transplanted into the field seven weeks after initial transferfrom culture media to potting mix, or after three weeks when theplantlet was produced from another plantlet in potting mix.

The composition of the M^(c)Cown Woody Plant medium used in thesestudies was as follows:

Micro elements CuSO₄•5H₂0 0.2 mg/l FeNaEDTA 36.70 mg/l H₃BO₃ 6.20 mg/lMnSO₄•H₂O 22.30 mg/l Na₂MoO₄•2H₂O 0.25 mg/l ZnSO₄•7H₂O 8.60 mg/l Macroelements CaCl₂ 72.50 mg/l Ca(NO₃)₂ 386.80 mg/l KH₂PO₄ 170.00 mg/l K₂SO₄990.00 mg/l MgSO₄ 180.54 mg/l NH₄NO₃ 400.00 mg/l Vitamins Glycine 2.00mg/l myo-Inositol 100.00 mg/l Nicotinic acid 0.50 mg/l Pyridoxine HCl0.50 mg/l Thiamine HCl 1.00 mg/l

The composition of Murashige and Skoog medium used in these studies wasas follows:

Ammonium nitrate 1,650 mg/l Boric acid 6.2 mg/l Calcium chloride 440mg/l Cobalt chloride 0.025 mg/l Magnesium sulfate 370 mg/l Cupricsulfate 0.025 mg/l Potassium phosphate 170 mg/l Ferrous sulfate 27.8mg/l Potassium nitrate 1,099 mg/l Manganese sulfate 22.3 mg/l Potassiumiodine 0.83 mg/l Sodium molybdate 0.25 mg/l Zinc sulfate 8.6 mg/lNa₂EDTA•2H₂O 37.2 mg/l i-Inositol 100 mg/l Niacin 0.5 mg/lPyridoxine•HCl 0.5 mg/l Thiamine•HCl 0.1 mg/l IAA 1 mg/l Kinetin 0.04mg/l Glycine 2.0 mg/l Edamine 1.0 g/l

EXAMPLE 4 Salix Spp Field Trial

Site trials were performed to determine the Salix species and growthregimes suitable for sites within the Lake Taupo catchments in NewZealand. Salix viminalis (a male clone) and Salix schwerinii‘Kinuyanagi’ (an infertile male clone) were selected as preferredspecies. These cultivars were initially selected on the basis thatcommercial nurseries considered them to be high yielding and resistantto insect pests such as sawfly. The trials were established using 20 to33 cm dormant stem cuttings planted at a stocking of ten to twelvethousand stem per hectare. Parameters that were evaluated for theoptimum biomass production included planting density, stake length, soilpreparation methods, fertilization regime, insect and weed management,and harvesting method.

The trial consisted of 32 plots (16 per species), testing sitepreparation of ripping compared with no ripping and cutting length.Cutting lengths of 20, 25 and 33 cm were tested. Weed control usingGardoprim® (Orion Crop Protection Ltd, Auckland, New Zealand) wasapplied to all plots. No fertilizer was applied due to the need tobenchmark soil and foliage analysis in the first year. The designatedmeasurement plot of forty trees was assessed in May 2006. Height of thedominant shoot, number of leaders greater than 50 cm from each cuttingand the number of live cuttings converted to a stocking (stems/ha) wasrecorded (Table 6).

As seen in Table 6, there were no significant differences in height andnumber of leader stems between Salix viminalis and Salix schwerinii‘Kinuyanagi’ one year after planting. Both species were well establishedon this specific site (light pumice based Taupo soil) independent of thesite preparation method. It should be noted that ripping would berequired if cuttings were planted mechanically. There was an increase inproductivity with 25 cm cutting as compared with the 20 cm cutting butfurther increases in cutting length (from 25 to 33) had no impact on thestocking rates and mean height of the dominant shoot.

TABLE 6 Site trial measurement after one year Mean Height Mean NumberStocking Variety (m) of Leaders (stems/ha) Species schwerinii 1.21 2.628,995 viminalis 1.18 2.78 9,403 Site preparation Ripped 1.30 2.84 8,909Unripped 1.11 2.81 10,006 Cutting length 20 1.14 2.51 8,856 25 1.23 2.859,876 33 1.25 3.11 9,641

The basic wood density, moisture content of one year old Salixschwerinii ‘Kinuyanagi’ and Salix viminalis were calculated (Table 7).Samples were collected for biomass analysis from four plants per plotswhere medium survival was recorded (2 plots per species).

TABLE 7 Biomass Analysis Species Wood density (kg/m³) Moisture (%) Sschwerinii 390 56.0 S. viminalis 384 55.5

While the present invention has been described with reference to thespecific embodiments thereof, it should be understood by those skilledin the art that various changes may be made and equivalents may besubstituted without departing from the true spirit and scope of theinvention. In addition, many modifications may be made to adapt aparticular situation, material, composition of matter, method, methodstep or steps, for use in practicing the present invention. All suchmodifications are intended to be within the scope of the claims appendedhereto.

All of the publications, patent applications and patents cited in thisapplication are herein incorporated by reference in their entirety tothe same extent as if each individual publication, patent application orpatent was specifically and individually indicated to be incorporated byreference in its entirety.

We claim:
 1. A method for extracting lignin from a lignocellulosic plantmaterial, comprising: (a) contacting lignocellulosic plant materialsolids with a continuous flow of an aqueous ethanol solution at anelevated temperature and at an elevated pressure, substantially in theabsence of an introduced acid catalyst, under conditions that promotedepletion of lignin from the lignocellulosic plant material solids andretention of hemicellulose sugars, xylose and cellulose in the plantmaterial solids to produce: i) a liquid mixture comprising ethanol andethanal-soluble lignin; and ii) treated plant material solids, whereinthe treated plant material solids are depleted of lignin and have a highconcentration of hemicellulose sugars and cellulose; (b) separating thetreated plant material solids from the liquid mixture; and (c)recovering the ethanol-soluble lignin from the liquid mixture.
 2. TheMethod of claim 1, wherein the lignocellulosic plant material is electedfrom the group consisting of: woody Or herbaceous materials,agricultural or forestry residues, and dedicated energy crops.
 3. Themethod of claim 1, wherein the lignocellulosic plant material isselected from the group consisting of: Salix, Poplar, Eucalyptus, switchgrass, miscanthus, sugar cane bagasse, soybean stover, corn stover, ricestraw, barley straw, wheat straw, corn fiber, wood fiber, andcombinations thereof.
 4. The method of claim 1, wherein the aqueousethanol solution comprises 50% to 80% ethanol.
 5. The method of claim 1,wherein the elevated temperature is between 170° C. and 210° C.
 6. Themethod of claim 1, wherein the elevated pressure is between 19-30 barg.7. The method of claim 1, additionally comprising drying thelignocellulosic plant material solids prior to contacting with theaqueous ethanol solution.
 8. The method of claim 1, wherein thelignocellulosic plant material solids are contacted with acounter-current flow of the aqueous ethanol solution.
 9. The method ofclaim 1, wherein the lignocellulosic plant material solids are contactedwith a co-current flow of the aqueous ethanol solution.
 10. The methodof claim 1, wherein the lignocellulosic plant material solids arecontacted with the aqueous ethanol solution in a screw contactordigester.
 11. The method of claim 1, wherein the lignocellulosic plantmaterial solids are contacted with the aqueous ethanol solution in acontinuous flow digester.
 12. The method of claim 1, wherein acontinuous flow of the lignocellulosic plant material solids iscontacted with a co-current or counter-current continuous flow of theaqueous ethanol solution.
 13. The method of claim 1, wherein aqueousethanol solution comprises about 70% ethanol and at least 28% of ligninpresent in the lignocellulosic plant material solids is solubilized. 14.An isolated lignin prepared by the method of claim
 1. 15. A method forextracting lignin from plant material, comprising: contacting plantmaterial solids with a continuous flow of an aqueous ethanol solution atan elevated temperature and at an elevated pressure, substantially inthe absence of an introduced acid catalyst, under conditions thatpromote depletion of lignin from the plant material solids and retentionof hemicellulose sugars, xylose and cellulose in the plant materialsolids to produce: i) a liquid mixture comprising ethanol andethanol-soluble lignin; and ii) treated plant material solids, whereinthe treated plant material solids are depleted of lignin and have a highconcentration of hemicellulose sugars and cellulose; separating thetreated plant material solids from the liquid mixture; and recoveringthe ethanol-soluble lignin from the liquid mixture, wherein the plantmaterial is from a plant selected from the group consisting of: Salixspecies and wheat.
 16. The method of claim 15, wherein the aqueousethanol solution comprises 50 to 80% ethanol.
 17. The method of claim15, wherein the elevated temperature is between 170° C. and 210° C. 18.The method of claim 15, wherein the elevated pressure is between 19-30barg.
 19. An isolated lignin prepared by the method of claim
 15. 20. Amethod for propagating plants of a Salix species, the method comprising:(a) culturing at least one shoot of a first plant selected from thegroup consisting of Salix species and collecting at least one cuttingfrom the shoot, wherein the cutting contains at least one node; and (b)cultivating the cutting in a comparison for a period of time sufficientto form a second plant, wherein the composition comprises benzyladenine,activated charcoal and at least one medium selected from the groupconsisting of: Murashige and Skoog medium and McCown Woody Plant medium.